Production of the metastatic phenotype with DNA from metastatic cells but not with oncogenic DNA

Although the tumorigenic phenotype can be transferred by transfecting DNA from a variety of human tumours into 3T3 mouse fibroblasts, the ability to transfer the metastatic phenotype in a similar manner in syngeneic animals is largely unknown. We now report that transfection of a rat mammary epithelial cell line Rama 37, which produces nonmetastasizing adenomatous tumours in syngeneic rats, with DNA from a metastasizing rat mammary cell line, Rama 800, yields some transfectants with reproducible metastatic capabilities. Nonspecific DNA or the oncogenes <sub>EJ-ras-1,</sub> or Polyoma Large T Antigen fail in this respect, although their transfectants often possess increased tumorigenic potential

Generation of metastatic variants by transfection of a nonmetastatic rat mammary epithelial cell line with DNA from a metastatic rat mammary cell line

Transfection of rat mammary (Rama) 37 epithelial cells, which yield nonmetastasizing adenomas in syngeneic Wistar-Furth rats, with HindlΠ-fragmented cellular DNA and the drug-resistance plasmids pSV2, gpí or <i>pSV2neo</i> yields drug-resistant transformants with a frequency of 10<sup>-4–</sup>10<sup>-5</sup>. Transformant cell lines transfected with the following, <i>pSV2gpt</i> alone, <i>pSV2gpt</i> and Rama 37 DNA, <i>pSV2gpt</i> and DNA from a metastasizing cell line Rama 800 (CT set), <i>pSVlneo</i> and salmon sperm DNA, pSV2neø and Rama 800 DNA (C set), all yield tumors when injected subcutaneously into syngeneic rats. A few transformants obtained by cotransfection with DNA from Rama 800 cells produce metastases in lungs and/or lymph nodes. The incidence of such metastases for two transfectants, termed CT4–41 and C18P, is significant at 20 and 24%, respectively, but only half (48%) that achieved with Rama 800 cells. Reintroduction into rats of cells cultured from a metastatic tumor of CT4–41 and of C18P, and from their lung or lymph node metastases, produces either a similar incidence (20–24%) or a significantly higher (48–52%) incidence of metastasis than that of the original transfectants. Cells cultured from nonmetastatic tumors fail to produce any metastatic lesions. When [<sup>32</sup>P]-labeled <i>gpt</i> or <i>neo</i> DNAs are hybridized to EcoRI-digested cellular DNA of the CT4–41 or C18P series of cell lines, tumors or metastases, <i>gpt</i> binds to one major fragment of 3,800 basepairs, and <i>neo</i> to two major fragments of 5,700 and 4,200 basepairs. The same cell lines produce hybridizing mRNAs of 1,500 and 1,900 bases for the CT4–41 series and 2,000–2,400 bases for the C18P series. It is suggested that transfection of DNA from the metastatic cells causes the nonmetastatic cells to become metastatic, in a genetically dominant manner, but additional steps are required for this process to become established and expressed at a level equivalent to that of the original, metastastasizing donor cells

Dedifferentiation of rat mammary myoepithelial-like cell lines after passage in vivo or cloning in vitro

Myoepithelial-like cell lines from normal mammary glands of neonatal Ludwig Wistar rats, rat mammary (Rama) 401 and Rama 704E, were injected into fat pads of syngeneic animals or were single-cell cloned in vitro. Rama 401 produced tumors that were predominantly composed of elongated cells, while the subclones of both cell lines yielded multilayered structures of elongated cells when grown on floating 0.3% collagen gels in vitro. Immunocytochemical analysis of histologic sections for markers of myoepithelial cells revealed that anti-actin-myosin and human keratin sera failed to stain the Rama 401 tumor cells or subclones of both cell lines on collagen gels, but both were stained with antilaminin serum. Immunofluorescent analysis of cultures of Rama 401 tumors showed that the resulting elongated cells failed to stain with antikeratin serum, but abundant staining was observed with antilaminin and antivimentin sera, as in the tumors. Ultrastructural analysis of the Rama 401 tumor cells identified intermediate junctions and extracellular basement membrane-like material in the vicinity of plasma membrane-associated pinocytotic vesicles, but neither true desmosomes nor myofilamental bundles were observed. Thus growth of rat mammary myoepithelial-like cells as tumors in syngeneic animals or as subclones in vitro can lead to selective loss of myofilaments and prekeratin-containing intermediate filaments. Similar relatively undifferentiated elongated cells may be responsible for some of the cellular heterogeneity observed in certain carcinogen-induced rat mammary tumors

Differential involvement of ERK1-2 and p38MAPK activation on Swiss 3T3 cell proliferation induced by prostaglandin F2α

Prostaglandin F2α (PGF2α) induces cyclin D1 expression and DNA synthesis in Swiss 3T3 cells. In order to assess which signaling mechanisms are implicated in these processes, we have used both a pharmacological approach and interfering mutants. We demonstrate that PGF2α induces extracellular-signal-regulated kinase (ERK1-2) and p38MAPK activation, and inhibition of any of these signaling pathways completely blocks PGF2α-stimulated DNA synthesis. We also show that ERK1-2, but not p38MAPK activation is required to induce cyclin D1 expression, strongly suggesting that the concerted action of cyclin D1 gene expression and other events are required to induce complete phosphorylation of retinoblastoma protein and S-phase entry in response to PGF2α. © 2006.Fil:Dekanty, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Giulianelli, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Coso, O.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Jimenez de Asua, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina